Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves
Abstract Background 1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosyn...
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| Format: | Article |
| Language: | English |
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BMC
2024-02-01
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| Series: | BMC Plant Biology |
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| Online Access: | https://doi.org/10.1186/s12870-024-04815-0 |
| _version_ | 1827328009078571008 |
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| author | Yangzhen Liao Wenmin Du Jingqiong Wan Jiahe Fan Jilan Pi Min Wu Yuan Wei Zhen Ouyang |
| author_facet | Yangzhen Liao Wenmin Du Jingqiong Wan Jiahe Fan Jilan Pi Min Wu Yuan Wei Zhen Ouyang |
| author_sort | Yangzhen Liao |
| collection | DOAJ |
| description | Abstract Background 1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosynthesis, its activity depends on the electron supply of NADPH-cytochrome P450 reductases (CPRs). However, the gene for MaCPRs in mulberry leaves remains unknown. Results In this study, we successfully cloned and functionally characterized two key genes, MaCPR1 and MaCPR2, based on the transcriptional profile of mulberry leaves. The MaCPR1 gene comprised 2064 bp, with its open reading frame (ORF) encoding 687 amino acids. The MaCPR2 gene comprised 2148 bp, and its ORF encoding 715 amino acids. The phylogenetic tree indicates that MaCPR1 and MaCPR2 belong to Class I and Class II, respectively. In vitro, we found that the recombinant enzymes MaCPR2 protein could reduce cytochrome c and ferricyanide using NADPH as an electron donor, while MaCPR1 did not. In yeast, heterologous co-expression indicates that MaCPR2 delivers electrons to MaC3'H hydroxylase, a key enzyme catalyzing the production of chlorogenic acid from 3-O-p-coumaroylquinic acid. Conclusions These findings highlight the orchestration of hydroxylation process mediated by MaCPR2 during the biosynthesis of secondary metabolite biosynthesis in mulberry leaves. These results provided a foundational understanding for fully elucidating the DNJ biosynthetic pathway within mulberry leaves. |
| first_indexed | 2024-03-07T15:10:45Z |
| format | Article |
| id | doaj.art-172bc6ef58404866b43c0e248211bd6b |
| institution | Directory Open Access Journal |
| issn | 1471-2229 |
| language | English |
| last_indexed | 2024-03-07T15:10:45Z |
| publishDate | 2024-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Plant Biology |
| spelling | doaj.art-172bc6ef58404866b43c0e248211bd6b2024-03-05T18:37:52ZengBMCBMC Plant Biology1471-22292024-02-0124111210.1186/s12870-024-04815-0Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leavesYangzhen Liao0Wenmin Du1Jingqiong Wan2Jiahe Fan3Jilan Pi4Min Wu5Yuan Wei6Zhen Ouyang7School of Food and Biological Engineering, Jiangsu UniversitySchool of Pharmacy, Jiangsu UniversitySchool of Pharmacy, Jiangsu UniversitySchool of Food and Biological Engineering, Jiangsu UniversitySchool of Pharmacy, Jiangsu UniversitySchool of Pharmacy, Jiangsu UniversitySchool of Pharmacy, Jiangsu UniversitySchool of Food and Biological Engineering, Jiangsu UniversityAbstract Background 1-Deoxynojirimycin (DNJ), the main active ingredient in mulberry leaves, with wide applications in the medicine and food industries due to its significant functions in lowering blood sugar, and lipids, and combating viral infections. Cytochrome P450 is a key enzyme for DNJ biosynthesis, its activity depends on the electron supply of NADPH-cytochrome P450 reductases (CPRs). However, the gene for MaCPRs in mulberry leaves remains unknown. Results In this study, we successfully cloned and functionally characterized two key genes, MaCPR1 and MaCPR2, based on the transcriptional profile of mulberry leaves. The MaCPR1 gene comprised 2064 bp, with its open reading frame (ORF) encoding 687 amino acids. The MaCPR2 gene comprised 2148 bp, and its ORF encoding 715 amino acids. The phylogenetic tree indicates that MaCPR1 and MaCPR2 belong to Class I and Class II, respectively. In vitro, we found that the recombinant enzymes MaCPR2 protein could reduce cytochrome c and ferricyanide using NADPH as an electron donor, while MaCPR1 did not. In yeast, heterologous co-expression indicates that MaCPR2 delivers electrons to MaC3'H hydroxylase, a key enzyme catalyzing the production of chlorogenic acid from 3-O-p-coumaroylquinic acid. Conclusions These findings highlight the orchestration of hydroxylation process mediated by MaCPR2 during the biosynthesis of secondary metabolite biosynthesis in mulberry leaves. These results provided a foundational understanding for fully elucidating the DNJ biosynthetic pathway within mulberry leaves.https://doi.org/10.1186/s12870-024-04815-0Morus alba L.NADPH-cytochrome P450 reductasesFunctional characterization |
| spellingShingle | Yangzhen Liao Wenmin Du Jingqiong Wan Jiahe Fan Jilan Pi Min Wu Yuan Wei Zhen Ouyang Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves BMC Plant Biology Morus alba L. NADPH-cytochrome P450 reductases Functional characterization |
| title | Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves |
| title_full | Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves |
| title_fullStr | Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves |
| title_full_unstemmed | Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves |
| title_short | Mining and functional characterization of NADPH-cytochrome P450 reductases of the DNJ biosynthetic pathway in mulberry leaves |
| title_sort | mining and functional characterization of nadph cytochrome p450 reductases of the dnj biosynthetic pathway in mulberry leaves |
| topic | Morus alba L. NADPH-cytochrome P450 reductases Functional characterization |
| url | https://doi.org/10.1186/s12870-024-04815-0 |
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